Role and localization of the glyoxylate cycle in the model microalga Chlamydomonas reinhardtii cultivated under day/night cycles

The ability of Chlamydomonas reinhardtii to grow under heterotrophic conditions, without light and solely with an exogeneous organic carbon source (acetate) in its cultivation medium, is one the particular features that have brought this green microalga to be considered as a model organism for the last 50 years, especially for studies on photosynthesis. Assimilation of acetate as an energy source is enabled by the glyoxylate cycle (GC), whose steps are quite similar to the ones of the Krebs cycle but comprises two specific enzymes, namely isocitrate lyase (ICL) and malate synthase (MAS). This cycle is well-known in other model organisms such as the yeast S. cerevisiae and the land plant A. thaliana but has been only poorly studied in Chlamydomonas since its discovery back in 1957, and more particularly when cells are cultivated under alternating light and dark phases, conditions under which it is thought to play a crucial role for the development of the alga. During this project, we started by precisely identifying the isoforms of the enzymes comprised in the GC in addition to their subcellular localisation. It came out that all of them are located inside the peroxisomes with the notable exception of ICL which is located in the cytosol. We then studied the evolution with time during two consecutives day/night cycles (12h/12h) of some of the main central carbon metabolism molecules which are tightly interconnected with the glyoxylate cycle. This was performed with control strains at first and then with a mutant strain deficient for the ICL gene, allowing us to observe that most of these metabolites accumulation profiles are influenced by the cell cycle and division rate, and that the GC is essential for the growth of C. reinhardtii under such alternate light conditions with acetate supplemented in the cultivation medium. Finally, in order to have a better comprehension of the cellular regulation mechanisms linked to these phenotypical observations, we performed a transcriptional analysis of data sampled from the same cultivation conditions using a method named “Surprisal Analysis”. Application of this original method enabled to show that control cells are highly influenced by acetate metabolism during day phase and by replication mechanisms during night phase while mutant cells, which are unable to divide, are markedly influenced by stress-related pathways during both phases but more particularly when light is turned off and they have therefore access to no source of energy to maintain their metabolism

Contribution to the study of mitochondrial metabolism and genome in Chlamydomonas reinhardtii

For the last decade, there has been growing concern about oil reserves depletion. Chlamydomonas reinhardtii, as well as other microalgae, has been investigated with a view to producing biofuel. But current production costs are still too high and impede the commercial implementation of the biofuel from microalgae strategy. Genetic improvement of C. reinhardtii is, thus, an area of immense interest in the worldwide scientific community. During this work, we studied a mutant for the isocitrate lyase (ICL) enzyme. It is the key enzyme of the glyoxylate cycle, which allows Chlamydomonas to use acetate as a carbon source to grow and develop, especially when cultivated in the dark. Identification and characterization of effects of this mutation could, thus, be helpful to better understand the interplays between ICL and other metabolic pathways. This study of the icl mutant highlights modifications of several cellular functions such as respiration, amino acids biosynthesis and stress responses. The second part of this work was devoted to the determination of mitochondrial transcription start site(s) in Chlamydomonas reinhardtii. Although mitochondrial genome transformation can be performed for several years, little is known about the mechanism of the mtDNA transcription in Chlamydomonas and especially about the promoter(s) used. In this work, we establish an almost functional protocol for transcription start site(s) identification and results suggest that multiple promoters might exist, which has never been observed before

Peroxisomal microbodies are at the crossroads of acetate assimilation in the green microalga Chlamydomonas reinhardtii

Lauersen KJ, Willamme R, Coosemans N, Joris M, Kruse O, Remacle C. Peroxisomal microbodies are at the crossroads of acetate assimilation in the green microalga Chlamydomonas reinhardtii. Algal Research. 2016;16:266-274

Peroxisomal microbodies are at the crossroads of acetate assimilation in the green microalga Chlamydomonas reinhardtii

The glyoxylate cycle is essential for growth on C2 compounds such as acetate. In this investigation, for the first time, we have elucidated the subcellular localization of the enzymes of the glyoxylate cycle in the green microalga Chlamydomonas reinhardtii. Acetyl-CoA synthase and malate dehydrogenase exist as multiple isoforms in this microalga, therefore, we first identified those implicated in the glyoxylate cycle based on the observation that lack of isocitrate lyase (ICL) in a previously identified icl deficient mutantwas correlatedwith specific loss of the other enzymes of the glyoxylate cycle. In this work, we determined that five of the six enzymes associated with the glyoxylate cycle were found to be within peroxisomal microbodies. Citrate synthase, aconitase, malate synthase, malate dehydrogenase, and acetyl-CoA synthase are located in peroxisomal microbodies while isocitrate lyase is cytosolic. Our findings implicate a key role for these cellular compartments in acetate assimilation for Chlamydomonas.Microbodies have only recently been discovered in C. reinhardtii and their existence had been previously debated. The isoform specific subcellular localization determined here suggests that peroxisomal microbodies should be considered in the design of metabolic models for carbon assimilation in C. reinhardtii

Metabolomic analysis of the green microalga Chlamydomonas reinhardtii cultivated under day/night conditions

peer reviewe

Lack of isocitrate lyase in Chlamydomonas leads to changes in carbon metabolism and in the response to oxidative stress under mixotrophic growth.

Isocitrate lyase is a key enzyme of the glyoxylate cycle. This cycle plays an essential role in cell growth on acetate, and is important for gluconeogenesis as it bypasses the two oxidative steps of the tricarboxylic acid (TCA) cycle in which CO2 is evolved. In this paper, a null icl mutant of the green microalga Chlamydomonas reinhardtii is described. Our data show that isocitrate lyase is required for growth in darkness on acetate (heterotrophic conditions), as well as for efficient growth in the light when acetate is supplied (mixotrophic conditions). Under these latter conditions, reduced acetate assimilation and concomitant reduced respiration occur, and biomass composition analysis reveals an increase in total fatty acid content, including neutral lipids and free fatty acids. Quantitative proteomic analysis by 14 N/15 N labelling was performed, and more than 1600 proteins were identified. These analyses reveal a strong decrease in the amounts of enzymes of the glyoxylate cycle and gluconeogenesis in parallel with a shift of the TCA cycle towards amino acid synthesis, accompanied by an increase in free amino acids. The decrease of the glyoxylate cycle and gluconeogenesis, as well as the decrease in enzymes involved in beta-oxidation of fatty acids in the icl mutant are probably major factors that contribute to remodelling of lipids in the icl mutant. These modifications are probably responsible for the elevation of the response to oxidative stress, with significantly augmented levels and activities of superoxide dismutase and ascorbate peroxidase, and increased resistance to paraquat

Metabolomic analysis of the green microalga Chlamydomonas reinhardtii cultivated under day/night conditions

peer reviewe